Dual Targeting of Antioxidant and Metabolic Enzymes to the Mitochondrion and the Apicoplast of Toxoplasma gondii

Toxoplasma gondii is an aerobic protozoan parasite that possesses mitochondrial antioxidant enzymes to safely dispose of oxygen radicals generated by cellular respiration and metabolism. As with most Apicomplexans, it also harbors a chloroplast-like organelle, the apicoplast, which hosts various biosynthetic pathways and requires antioxidant protection. Most apicoplast-resident proteins are encoded in the nuclear genome and are targeted to the organelle via a bipartite N-terminal targeting sequence. We show here that two antioxidant enzymes—a superoxide dismutase (TgSOD2) and a thioredoxin-dependent peroxidase (TgTPX1/2)—and an aconitase are dually targeted to both the apicoplast and the mitochondrion of T. gondii. In the case of TgSOD2, our results indicate that a single gene product is bimodally targeted due to an inconspicuous variation within the putative signal peptide of the organellar protein, which significantly alters its subcellular localization. Dual organellar targeting of proteins might occur frequently in Apicomplexans to serve important biological functions such as antioxidant protection and carbon metabolism

Plastid targeting and the pyruvate dehydrogenase complex in the malaria parasite Plasmodium falciparum

Typescript (photocopy)Thesis (PhD) -- University of Melbourne, School of Botany, 2003Includes bibliographical references (leaves 167-182)CD-ROM content not included in digitised versionThe phylum Apicomplexa consists of obligate parasites that cause severe diseases in humans and livestock. The genera Plasmodium, Toxoplasma and Eimeria, for example, are the causative agents of malaria, toxoplasmosis, and coccidiosis. Apicomplexan parasites contain a relict plastid which has been termed �apicoplast�. Chapter 1 of this thesis presents an extensive literature review covering the evolutionary origin of the apicoplast, its division during the cell cycle, protein targeting and import, its metabolic function, and the effect of drugs on this organelle. Thus, it is argued that the apicoplast originated from the engulfment of a once free-living organism of the red algal lineage. Differences in organellar division between the apicomplexan plastid and chloroplasts of plants are highlighted. The review describes how the vast majority of the apicoplast proteome is encoded in the nuclear genome, and how the products of these nuclear genes are post-translationally targeted to the organelle via the secretory pathway courtesy of a bipartite N-terminal leader sequence. Finally, it summarises what is known to date about the metabolic pathways located within the apicoplast including fatty acid, isoprenoid and heme synthesis, and reviews the action of compounds that inhibit these functions. A study exploring the particular amino acid characteristics of apicoplast-targeting transit peptides from the malaria parasite Plasmodium falciparum is presented in Chapter 2. A novel algorithm (�PlasmoAP�) recognising this particular amino acid bias was developed which � in conjunction with the existing bioinformatic tool SignalP � was able to distinguish between apicoplast-targeted and non-apicoplast proteins encoded in the nuclear genome of P. falciparum. Site-directed mutagenesis altering charge-related amino acid features in a model transit peptide severely disrupted organellar targeting in vivo, confirming the biological significance of features embedded in PlasmoAP. In addition, putative Hsp70- or DnaK-binding sites are shown to be abundant in plasmodial plastid transit peptides, and the removal of predicted Hsp70-binding sites from a plasmodial transit peptide led to disruption of transit peptide function in vivo. Finally, the high AT-content of P. falciparum DNA is presented as a major driving force shaping the particular amino acid composition observed in plasmodial transit peptides. The pyruvate dehydrogenase complex (PDHC) is one member of a family of 2-oxo acid dehydrogenase complexes (ODHCs), and a concise introductory review in Chapter 3 provides an overview of these enormous multi-subunit enzyme complexes that occupy central roles in the metabolism of mitochondria and plastids. Several ODHCs were identified in the genomes of five Plasmodium species and of Toxoplasma gondii, indicating that these parasites contain one pyruvate dehydrogenase complex (PDHC) in the apicoplast, as well as one ?-ketoglutarate dehydrogenase complex (KGDHC) and one branched-chain ?-ketoacid dehydrogenase complex (BCKDHC) in the mitochondrion. The four genes encoding a complete PDHC in P. falciparum were confirmed through sequencing of cDNA clones, and 10 introns were identified in the E2 subunit sequence. Phylogenetic analyses and the apparent presence of bipartite N-terminal leaders are presented as strong circumstantial evidence arguing that the P. falciparum PDHC is located in the apicoplast. Chapter 4 describes the recombinant expression of the constituent subunits of the PDHC identified in P. falciparum. Primary sequence comparisons and high enzymatic activity measured for the recombinantly expressed catalytic domain of the PDHC subunit E2 indicate that the parasite PDHC genes encode functional enzymes. Polyclonal antibodies directed against the plasmodial PDHC subunits E1?, E1?, and E2 were generated in rabbits, and Western blot data suggests that these proteins are expressed in vivo. The thesis closes by drawing overall conclusions from the PhD research presented and by providing an outlook for its future implications and ongoing related work

Molecular and functional aspects of parasite invasion

Apicomplexan parasites have evolved an efficient mechanism to gain entry into non-phagocytic cells, hence challenging their hosts by the establishment of infection in immuno-privileged tissues. Gliding motility is a prerequisite for the invasive stage of most apicomplexans, allowing them to migrate across tissues, and actively invade and egress host cells. In the late 1960s, detailed morphological studies revealed that motile apicomplexans share an elaborate architecture comprising a subpellicular cytoskeleton and apical organelles. Since 1993, the development of technologies for transient and stable transfection have provided powerful tools with which to identify gene products associated with these structures and organelles, as well as to understand their functions. In combination with access to several parasite genomes, it is now possible to compare and contrast the strategies and molecular machines that have been selectively designed by distinct life stages within a species, or by different apicomplexan species, to optimize infection

New insights into myosin evolution and classification

Myosins are eukaryotic actin-dependent molecular motors important for a broad range of functions like muscle contraction, vision, hearing, cell motility, and host cell invasion of apicomplexan parasites. Myosin heavy chains consist of distinct head, neck, and tail domains and have previously been categorized into 18 different classes based on phylogenetic analysis of their conserved heads. Here we describe a comprehensive phylogenetic examination of many previously unclassified myosins, with particular emphasis on sequences from apicomplexan and other chromalveolate protists including the model organism Toxoplasma, the malaria parasite Plasmodium, and the ciliate Tetrahymena. Using different phylogenetic inference methods and taking protein domain architectures, specific amino acid polymorphisms, and organismal distribution into account, we demonstrate a hitherto unrecognized common origin for ciliate and apicomplexan class XIV myosins. Our data also suggest common origins for some apicomplexan myosins and class VI, for classes II and XVIII, for classes XII and XV, and for some microsporidian myosins and class V, thereby reconciling evolutionary history and myosin structure in several cases and corroborating the common coevolution of myosin head, neck, and tail domains. Six novel myosin classes are established to accommodate sequences from chordate metazoans (class XIX), insects (class XX), kinetoplastids (class XXI), and apicomplexans and diatom algae (classes XXII, XXIII, and XXIV). These myosin (sub)classes include sequences with protein domains (FYVE, WW, UBA, ATS1-like, and WD40) previously unknown to be associated with myosin motors. Regarding the apicomplexan "myosome," we significantly update class XIV classification, propose a systematic naming convention, and discuss possible functions in these parasites

Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNAMet formylation in Apicomplexa

Apicomplexans possess three translationally active compartments: the cytosol, a single tubular mitochondrion, and a vestigial plastid organelle called apicoplast. Mitochondrion and apicoplast are of bacterial evolutionary origin and therefore depend on a bacterial-like translation machinery. The minimal mitochondrial genome contains only three ORFs, and in Toxoplasma gondii the absence of mitochondrial tRNA genes is compensated for by the import of cytosolic eukaryotic tRNAs. Although all compartments require a complete set of charged tRNAs, the apicomplexan nuclear genomes do not hold sufficient aminoacyl-tRNA synthetase (aaRSs) genes to be targeted individually to each compartment. This study reveals that aaRSs are either cytosolic, apicoplastic or shared between the two compartments by dual targeting but are absent from the mitochondrion. Consequently, tRNAs are very likely imported in their aminoacylated form. Furthermore, the unexpected absence of tRNA<sup>Met</sup> formyltransferase and peptide deformylase implies that the requirement for a specialized formylmethionyl-tRNA<sup>Met</sup> for translation initiation is bypassed in the mitochondrion of Apicomplexa

Unusual Anchor of a Motor Complex (MyoD-MLC2) to the Plasma Membrane of Toxoplasma gondii

Toxoplasma gondii possesses 11 rather atypical myosin heavy chains. The only myosin light chain described to date is MLC1, associated with myosin A, and contributing to gliding motility. In this study, we examined the repertoire of calmodulin-like proteins in Apicomplexans, identified six putative myosin light chains and determined their subcellular localization in T. gondii and Plasmodium falciparum. MLC2, only found in coccidians, is associated with myosin D via its calmodulin (CaM)-like domain and anchored to the plasma membrane of T. gondii via its N-terminal extension. Molecular modeling suggests that the MyoD-MLC2 complex is more compact than the reported structure of Plasmodium MyoA-myosin A tail-interacting protein (MTIP) complex. Anchorage of this MLC2 to the plasma membrane is likely governed by palmitoylation